An Optimized Protocol for the Generation of Alveolospheres from Wild-Type Mice

Organoid models have become an integral part of the research methodology in the lung field. These systems allow for the study of progenitor and stem cell self-renewal, self-organization, and differentiation. Distinct models of lung organoids mimicking various anatomical regions of mature lungs have emerged in parallel to the increased gain of knowledge regarding epithelial stem and progenitor cell populations and the corresponding mesenchymal cells that populate the in vivo niche. In the distal lung, type 2 alveolar epithelial cells (AEC2s) represent a stem cell population that is engaged in regenerative mechanisms in response to various insults. These cells self-renew and give rise to AEC1s that carry out gas exchange. Multiple experimental protocols allowing the generation of alveolar organoids, or alveolospheres, from murine lungs have been described. Among the drawbacks have been the requirement of transgenic mice allowing the isolation of AEC2s with high viability and purity, and the occasional emergence of bronchiolar and bronchioalveolar organoids. Here, we provide a refined gating strategy and an optimized protocol for the generation of alveolospheres from wild-type mice. Our approach not only overcomes the need for transgenic mice to generate such organoids, but also yields a pure culture of alveolospheres that is devoid of bronchiolar and bronchioalveolar organoids. Our protocol contributes to the standardization of this important research tool.


Introduction
Organoids are three-dimensional (3D) structures that are representative of the structure, cellular morphology, and certain functional features of the tissue or organ of interest.In recent years, murine and human organoids have emerged as powerful tools for studying organ development, cellular interaction, drug screening, and regenerative mechanisms.Due to their unlimited capacity to proliferate and differentiate, adult stem and progenitor cells have been widely used to develop organoids for research purposes [1,2].
In the context of pulmonary research, several murine organoid systems have been developed to create airway and/or alveolar structures resembling various aspects of lung organogenesis, homeostasis, and regeneration [3].For instance, tracheal organoids, or tracheospheres, with a visible lumen can be grown from basal stem cells even in the absence of stromal cells or other non-basal cells [4].Airway organoids, or bronchiolospheres, can be generated by co-culturing club cells with airway smooth muscle cells (ASMCs) or repair-supportive mesenchymal cells (RSMCs), thus mimicking the intercellular communication that occurs during airway epithelial regeneration in vivo following naphthalene injury [5][6][7][8].Such bronchiolospheres can also be generated by culturing club cells with [CD45− CD31− EpCAM− Sca-1+] (non-leukocytic, non-endothelial, non-epithelial, stem cell antigen-1-expressing) resident mesenchymal cells (rMCs) [5].The most comprehensive system so far is the bronchioalveolar lung organoid (BALO) model, which relies on co-culturing bronchioalveolar stem cells (BASCs) and rMCs, resulting in a highly sophisticated mimicry of the bronchioalveolar region of the mature mouse lung [7,9].
Type 2 alveolar epithelial cells (AEC2s) represent a stem cell population in the alveolar compartment of the lung [10].These cells self-renew and regenerate this compartment by differentiating into type 1 alveolar epithelial cells (AEC1s) that are responsible for gas exchange, eventually via intermediate cell types [11][12][13].Although the heterogeneous nature of AEC2s was not obvious initially, emerging research suggests that there are indeed AEC2 subsets that display varying stem-cell features.For example, WNT-responsive AEC2s (expressing axis inhibition protein 2 or AXIN2), termed alveolar epithelial progenitors (AEPs), have been shown to represent a stable lineage during homeostasis, and are capable of rapid expansion following acute lung injury [14].Another subpopulation of AEC2s is enriched in programmed cell death 1 ligand 1 (PD-L1 or CD274) expression and was dubbed "injury-associated alveolar progenitors" (IAAPs).These cells replenish the mature AEC2 pool upon alveolar injury [15,16].
Isolation of AEC2s by FACS for alveolosphere cultures has so far mostly relied on the use of genetically modified mice (Sftpc Cre-ERT2/+ ; tdTomato flox ) that allow labeling of surfactant protein C-positive (SFTPC+) AEC2s.Such cells can also be filtered through the additional use of Lysotracker, a dye that stains lysosomes and therefore detects lamellar bodies that are abundant in AEC2s [17].A population of rMCs that expresses fibroblast growth factor 10 (FGF10) and contains neutral lipids (therefore possessing a lipofibroblastlike phenotype) promotes the formation of AEC2-derived alveolospheres containing AEC2s and AEC1s [18,19].Alveolospheres had been initially described by co-culturing lineagelabeled AEC2s with platelet-derived growth factor receptor alpha (PDGFRα)-expressing mesenchymal cells, also termed type 2-associated stromal cells (TASCs), that display lipofibroblast characteristics [10,20].The co-culture assay using Sca-1+ mesenchymal cells and lung epithelial progenitor cells had already been developed [21].
To overcome the need for transgenic mice for the generation of alveolospheres, we hereby provide a detailed protocol for culturing such organoids starting with wild-type (WT) mouse lungs.We validated various aspects of our approach including colony-forming efficiency (CFE), alveolosphere diameter, and cellular differentiation by comparison with alveolospheres obtained from Sftpc Cre-ERT2/+ ; tdTomato flox mice.Our protocol serves as a resource for the lung stem-cell research community interested in AEC2 self-renewal and differentiation, and interaction with mesenchymal niche cells.

Mice and Tamoxifen Administration
Mice between 8 and 12 weeks of age were used and maintained on the C57BL/6 background.Male wild-type (WT) mice were purchased from Charles River (Strain code 632, Wilmington, MA, USA).Sftpc tm1(Cre/ERT2,rtTA)Hap (or simply Sftpc Cre-ERT2 ) mice were previously described [22], and Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze (or simply tdTomato flox ) mice were purchased from the Jackson Laboratory (stock number 007905, Bar Harbor, ME, USA).The two lines were bred to generate Sftpc Cre-ERT2/+ ; tdTomato flox mice.Tamoxifen powder (Sigma-Aldrich, T5648, St. Louis, MO, USA) was dissolved in corn oil (Sigma-Aldrich, C8267) and injected intraperitoneally three times at a dose of 0.25 mg/g body weight per injection.Mice were kept under specific pathogen-free (SPF) conditions with unlimited access to food and water.

Induction of Cre Activity in Ex Vivo Alveolosphere Cultures
To induce Cre-mediated recombination ex vivo, organoid medium containing 1 µM of 4-Hydroxytamoxifen (4-OHT) (Merck, H6278, Darmstadt, Germany) was added to the organoid cultures on day 1.After 24 h, the organoid medium containing 4-OHT was removed, and the organoid inserts were washed twice with 1X PBS.Then, the inserts were placed on a new 24-well plate and alveolospheres were cultured in organoid medium until day 16.

Whole-Mount Immunofluorescence
On day 16 of culture, alveolospheres were fixed with 4% paraformaldehyde (PFA) (Merck, 104005) for 20 min at RT, then washed three times with 1X PBS.Fixed alveolospheres were blocked and permeabilized with blocking buffer (5% serum, 0.5% Triton-X-100 in 1X PBS) overnight at 4 • C. The serum was chosen from the same species as the host of the secondary antibody.For immunofluorescence, alveolospheres were incubated with primary antibodies in a blocking buffer overnight at 4 • C. The next day, washing buffer (2% serum, 0.3% Triton-X-100 in 1X PBS) was added 3 times for 10-15 min, and secondary antibodies were added overnight at 4 • C. To stain nuclei, the NucBlue™ Fixed Cell ReadyProbes™ Reagent (Thermo Fisher) was utilized.Lastly, stained alveolospheres were washed three times with washing buffer for 10-15 min.The Matrigel containing the organoids was cut out of the insert with a scalpel.The sample was placed on a slide and covered with 5-6 drops of fructose-glycerol clearing solution (60% glycerol, 2.5 M fructose) [23].A coverslip was placed on the sample and after drying for 1 day at 4 • C in the dark, imaging was carried out using an SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) controlled by LAS X software (version 3.5.7).For capturing the confocal z-stacks, we used 20x0.75NAair and 63xW 1.2NA objectives, while the scan settings were kept the same across all acquisitions.A list of primary and secondary antibodies can be found in Table 1.
Table 1.List of antibodies used for immunofluorescence.

Image Analysis
The acquired confocal images were cleared computationally, using the Lightning module of LAS X software.Subsequently, for the maximum intensity projection and quantification of the z-stack, we used the ImageJ/Fiji software [24].With Fiji, every projected image was thresholded using the default integrated algorithm.Then, the area occupied by the positive signal (of each marker) as well as the total organoid area were measured.The final area ratio of each marker was calculated by dividing the marker's area by the organoid's area.

RNA Extraction and Gene Expression Analysis
RNA was extracted from alveolosphere cultures using the RNeasy micro kit (Qiagen, 74004, Hilden, Germany).cDNA synthesis was performed using the Quantitect reverse transcription kit (Qiagen, 205311) according to the manufacturer's instructions.Quantitative real-time PCR (qPCR) was performed using a PowerUp SYBR green master mix (Thermo Fisher Scientific, A25742) and a LightCycler 480 II machine (Roche Applied Science, Penzberg, Germany).Gene expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and presented as fold change relative to day 8 of culture.Primer sequences are shown in Table 2. Bulk RNA sequencing was carried out according to standard procedures.For genome-wide analysis of gene expression, RNA sequencing libraries from polyadenylated mRNA were generated and sequenced using Illumina NovaSeq 6000 at the Institute for Lung Health (ILH)-Genomics and Bioinformatics at the Justus-Liebig University (JLU) Giessen (Germany).
Table 2. Primer sequences used for qPCR.

Gene Name
Forward Primer Reverse Primer

Statistical Analysis
The number of biological replicates is provided in the corresponding figure legends.All data are presented as mean ± standard error of the mean (SEM).Student's t test (unpaired, two tailed) was used to compare the means of two groups and One-way ANOVA was used to compare more than two groups.The differences in the means were considered statistically significant when the p value was less than 0.05.Data were assembled and analyzed using GraphPad Prism software, version 9.5.1 (528) (GraphPad Software, Boston, MA, USA).
Gating for total tdTom+ cells (tdTom low and tdTom high ) showed that 91.4% ± 2.1% of these cells were contained in the EpCAM low population compared to traces in EpCAM high cells (Figure 1d).Gating for tdTom high and tdTom low populations showed similar results (Figure 1e,f).Further analysis of the EpCAM low population showed that 95% ± 0.42% of these cells were tdTom+, with the majority being tdTom high (64.1% ± 4.5%) and a minor proportion of tdTom low (30.2% ± 4.7%) (Figure 1g).On the other hand, the EpCAM high population showed lower proportions of tdTom+ cells (Figure 1h).Therefore, the EpCAM low population was confirmed to be enriched with AEC2s.

The EpCAM low Population Robustly Generates Alveolospheres and It Is Comparable with Sorted Lineage-Traced AEC2s
The data described above clearly show that gating for CD45− CD31− EpCAM low cells in WT lungs allows for the sorting of an AEC population that predominantly consists of AEC2s.The finding that lineage-traced AEC2s essentially belong to the EpCAM low population (Figure 1d-f) strongly supports this conclusion.We therefore tested the ability of EpCAM low cells to generate alveolospheres (WT approach) and compared the results to the TG approach.As expected, all alveolospheres in the TG approach cells were tdTom+ (Figure 2h).Both approaches were similarly efficient in generating alveolospheres (WT CFE 3.14% ± 0.23% vs. TG CFE 2.55% ± 0.27%) (Figure 2i) with comparable alveolosphere diameter (139.37 µm ± 6.86 µm for WT vs. 130.57µm ± 5.01 µm for TG) (Figure 2j).Gene expression analysis was then carried out on WT and TG alveolospheres on day 8 (when AECs start to form cysts) and day 16 (when alveolospheres acquire a complex morphology and contain differentiated AEC2s and AEC1s).The results showed significant upregulation of the AEC2 marker Sftpc (Figure 2k,m) and the AEC1 marker Ager (Figure 2l,n) in both conditions.
To further demonstrate that the alveolospheres generated using the WT approach are comparable to those generated using the TG approach, we carried out RNA-seq on isolated WT AECs, tdTom+ cells, and the corresponding organoids (Figure 4a).The full list of genes is shown in Supplementary Table S1.Notably, the same gene sets are modulated during organoid formation regardless of whether the starting material is tdTom+ cells or WT AECs (Figure 4b,c).Moreover, a comparison of AEC2 and AEC1 markers showed no difference between the two groups (Figure 4d,e), confirming that the cell population isolated using our WT EpCAM low approach is very similar to the tdTom+ population isolated from transgenic animals.We also found that the AEC1 signature is upregulated in alveolospheres compared with isolated AEC2s, while the AEC2 signature is diluted in alveolospheres (Figure 4d,e), which is expected given the emergence of AEC1s and the abundance of mesenchymal cells in the alveolospheres but not in purified AEC2s.Pathway analysis of the top genes differentially regulated between alveolospheres and AEC2s was related to ECM organization, elastic fibers, collagen biosynthesis, and cell motility (Figure 4f).

Stimulation with Recombinant Murine IL-1β Alters Alveolosphere Formation
It has been reported that IL-1β treatment leads to enlarged alveolospheres that feature accumulation of the KRT8+ intermediate cell state [12]; therefore, recombinant IL-1β was added to WT cultures as another proof of concept that such organoids behave similarly to those obtained using the TG approach (Figure 5a).After 16 days of culture, organoids clearly showed a cyst-like morphology rather than a typical alveolosphere morphology in the IL-1β group (Figure 5b), which was also reflected by the quantification of organoid diameter (285.08 µm ± 11.6 µm vs. 145.04µm ± 9.16 µm) (Figure 5d).Analysis of the CFE did not reveal a difference between the two groups (Figure 5c).Notably, immunofluorescence showed a significant increase in the coverage of the KRT8 signal relative to the overall alveolosphere area upon IL-1β treatment (0.36 ± 0.05 vs. 0.16 ± 0.05) (Figure 5e,f).These data indicate that our WT approach can recapitulate previous findings attained using the TG approach.
animals.We also found that the AEC1 signature is upregulated in alveolospheres compared with isolated AEC2s, while the AEC2 signature is diluted in alveolospheres (Figure 4d,e), which is expected given the emergence of AEC1s and the abundance of mesenchymal cells in the alveolospheres but not in purified AEC2s.Pathway analysis of the top genes differentially regulated between alveolospheres and AEC2s was related to ECM organization, elastic fibers, collagen biosynthesis, and cell motility (Figure 4f).  group (Figure 5b), which was also reflected by the quantification of organoid diameter (285.08 µm ± 11.6 µm vs. 145.04µm ± 9.16 µm) (Figure 5d).Analysis of the CFE did not reveal a difference between the two groups (Figure 5c).Notably, immunofluorescence showed a significant increase in the coverage of the KRT8 signal relative to the overall alveolosphere area upon IL-1β treatment (0.36 ± 0.05 vs. 0.16 ± 0.05) (Figure 5e,f).These data indicate that our WT approach can recapitulate previous findings attained using the TG approach.

Discussion
Over the past decade, lung organoids have emerged as important models to investigate morphogenic and regenerative aspects of lung biology and disease such as assessing epithelial stem/progenitor cell self-renewal, self-organization, and differentiation, as well as the supportive potential (or niche activity) of corresponding mesenchymal cells.These organoid systems mimic various anatomical regions of the mammalian lung and are therefore dubbed tracheospheres, bronchiolospheres, bronchioalveolar organoids, and alveolospheres [3,[25][26][27][28].As with any culture assay, there is a strong need for standardization of the operating procedure to maximize data quality, robustness, and reproducibility.
One of the pioneering studies in the field is that of McQualter et al., who demonstrated the ability of lung EpCAM+ cells to form airway, alveolar, and mixed organoids when co-cultured with EpCAM− Sca-1+ mesenchymal cells in Matrigel [21].The generation of organoids from murine and human AEC2s was later described, where the co-culture of AEC2s with PDGFRα+ mesenchymal cells or TASCs, a population that includes lipofibroblasts, led to the formation of alveolospheres containing AEC2s and AEC1s [10,20].Alveolosphere cultures were also used to characterize the WNT-responsive fraction of AEC2s in the murine lung and investigate alveolar niche interactions.Such an AEC2 subset displayed superior alveolosphere formation capacity and responds to WNT and FGF stimulation [14,29,30].Another AEC2 subset that acts as a quiescent repertoire for mature AEC2s is the IAAPs, which do not show robust alveolosphere formation capacity but are important for replenishing the bona fide AEC2 pool [15,16,31,32].
WNT and FGF signaling have been established as critical determinants of the AEC2 cell fate by independent studies [7,31,[33][34][35][36].The mesenchymal niche can be fractionated into AXIN2+ (a WNT-responsive population enriched with Fgf7 expression) and FGF10+ subsets.Both fractions show lipofibroblast features including proximity to AEC2s and neutral lipid content [18,37].How distinct these two subsets are, especially in terms of in vivo function during homeostasis, repair, and regeneration, remains to be seen.A third niche population is represented by WNT ligand-producing leucine-rich repeat-containing G protein-coupled receptor-5-positive (LGR5+) fibroblasts that have shown the ability to support alveolosphere formation [7].In the current protocol, the bulk rMC population was used to achieve a simple and universal protocol to generate alveolospheres from WT mice without the need for transgenic animals or discriminating between rMC subsets.Table 3 highlights the main features of some of the published protocols to generate murine alveolospheres.The emerging picture is that (1) most of the studies listed below relied on the use of transgenic mice to generate alveolospheres, (2) a starting material of 5000 AEC2s combined with mesenchymal cells at a ratio of 1:10 and later mixed with Matrigel (1:1) is the most used approach, and (3) the data show varying CFE values even when using similar protocols.

Information not available
Protocol to generate feeder-free alveolospheres ** * Experiments were also conducted with LGR6+ cells but predominantly yielded bronchiolar organoids rather than alveolospheres.** AEC1 differentiation was not achieved under these conditions.

Conclusions
Our protocol thus allows robust and reproducible generation of murine alveolsopheres using the EpCAM low gating strategy, which represents a refinement of previous EpCAM gating strategies and avoids lumping EpCAM high with EpCAM low cells.As club cells and BASCs are not found in the EpCAM low fraction [9,19], our approach minimizes contamination with bronchiolar and bronchioalveolar organoids, allowing to predominantly obtain alveolospheres.We believe that our protocol represents a step toward the standardization of this important research tool.
Finally, we were able to reproduce the effect of IL-1β on alveolosphere formation using our WT approach.The accumulation of KRT8+ transitional cells was evident based on whole-mount immunofluorescence data.The involvement of these and other transitional AEC2 populations in various respiratory diseases has gained tremendous interest in recent years [11,13] and further research is warranted to fully understand this phenomenon, decipher how to convert such dysplastic events into healthy scarless regeneration, and ultimately reconstitute the alveolar stem cell niche and restore optimal gas exchange in the diseased lung.

Figure 2 .
Figure 2. Generation of alveolospheres from WT and TG mouse lungs.(a) Experimental design for co-culturing WT or TG AEC2s with rMCs and subsequent analysis.(b) Gating strategy for sorting AEC2s (EpCAM low ) from WT mouse lungs.(c) Quantification of AEC1s and AEC2s in the EpCAM low population.(d) Gating strategy for identifying AEC2s (EpCAM low ) in TG mouse lungs.(e) Quantification of AEC1s and AEC2s in the EpCAM low population.(f) Gating strategy to select EpCAM low cells from the tdTom high population.(g) Quantification of AEC1s and AEC2s in the EpCAM low fraction of the tdTom high population.(h) Tile scans of representative wells for WT and TG alveolosphere cultures.(i,j) Quantification of CFE and alveolosphere diameter.(k-n) qPCR for alveolar epithelial marker genes on days 8 and 16 of culture.(c,e,g) Each dot represents one lung; (i,j) each dot represents one culture well; (k-n) each dot represents two pooled culture wells.** p < 0.01; *** p < 0.001; **** p < 0.0001; ns: not significant.AECs: alveolar epithelial cells; CFE: colony-forming efficiency; rMCs: resident mesenchymal cells; TG: transgenic; WT: wild type.Scale bars: 1 mm.

Figure 2 .
Figure 2. Generation of alveolospheres from WT and TG mouse lungs.(a) Experimental design for co-culturing WT or TG AEC2s with rMCs and subsequent analysis.(b) Gating strategy for sorting AEC2s (EpCAM low ) from WT mouse lungs.(c) Quantification of AEC1s and AEC2s in the EpCAM low population.(d) Gating strategy for identifying AEC2s (EpCAM low ) in TG mouse lungs.(e) Quantification of AEC1s and AEC2s in the EpCAM low population.(f) Gating strategy to select EpCAM low cells from the tdTom high population.(g) Quantification of AEC1s and AEC2s in the EpCAM low fraction of the tdTom high population.(h) Tile scans of representative wells for WT and TG alveolosphere cultures.(i,j) Quantification of CFE and alveolosphere diameter.(k-n) qPCR for alveolar epithelial marker genes on days 8 and 16 of culture.(c,e,g) Each dot represents one lung; (i,j) each dot represents one culture well; (k-n) each dot represents two pooled culture wells.** p < 0.01; *** p < 0.001; **** p < 0.0001; ns: not significant.AECs: alveolar epithelial cells; CFE: colony-forming efficiency; rMCs: resident mesenchymal cells; TG: transgenic; WT: wild type.Scale bars: 1 mm.

Cells 2024 , 16 Figure 3 .
Figure 3. Alveolospheres derive from SFTPC+ AEC2s and contain both AEC1s and AEC2s.(a,b) Experimental design for co-culturing uninduced TG AEC2s with WT rMCs and ex vivo induction with 4-OHT.(c,d) Gating strategy for sorting WT rMCs and tdTom− EpCAM low cells from uninduced TG mouse lungs.(e,f) Tile scans of representative wells on days 2 and 16.The dashed boxes are magnified in the right panels.The tdTom signal is shown in red.(g) Tile scan of a day 16 culture well in the absence of 4-OHT.(h) Quantification of the percentage of tdTom+ alveolospheres.(i) Representative images of the maximum intensity projection images of individual organoids from the indicated conditions stained for SFTPC and AGER.Note the presence of the tdTom+ signal in the TG and 4-OHT (TG) conditions but not the WT condition.(j,k) Quantification of the ratio of the SFTPC and AGER signals to alveolosphere area.(h) Each dot represents one culture well; (j,k) each dot represents one organoid.4-OHT: 4-hydroxytamoxifen; AECs: alveolar epithelial cells; ns: not significant; rMCs: resident mesenchymal cells; TG: transgenic; WT: wild type.Scale bars: (e-g) 1 mm; (i) 50 µm.

Figure 3 .
Figure 3. Alveolospheres derive from SFTPC+ AEC2s and contain both AEC1s and AEC2s.(a,b) Experimental design for co-culturing uninduced TG AEC2s with WT rMCs and ex vivo induction with 4-OHT.(c,d) Gating strategy for sorting WT rMCs and tdTom− EpCAM low cells from uninduced TG mouse lungs.(e,f) Tile scans of representative wells on days 2 and 16.The dashed boxes are magnified in the right panels.The tdTom signal is shown in red.(g) Tile scan of a day 16

Figure 4 .
Figure 4. Resemblance between the WT and TG approaches at the transcriptomic level.(a) Experimental design.(b) Scatter plot showing the correlation between the WT and TG approaches.(c) Heatmap showing the top 2,000 differentially expressed genes between alveolospheres and sorted cells.(d,e) Heatmaps showing selected AEC2 and AEC1 marker genes in various samples.(f) Reactome pathway analysis of the top differentially expressed genes in alveolospheres versus sorted cells.Pathway enrichment was calculated by multiplying −log10(p value) by +1 (in case of upregulation) or −1 (in case of downregulation).Alv.: alveolospheres; rMCs: resident mesenchymal cells; TG: transgenic; WT: wild type.

Figure 4 .
Figure 4. Resemblance between the WT and TG approaches at the transcriptomic level.(a) Experimental design.(b) Scatter plot showing the correlation between the WT and TG approaches.(c) Heatmap showing the top 2000 differentially expressed genes between alveolospheres and sorted cells.(d,e) Heatmaps showing selected AEC2 and AEC1 marker genes in various samples.(f) Reactome pathway analysis of the top differentially expressed genes in alveolospheres versus sorted cells.Pathway enrichment was calculated by multiplying −log 10 (p value) by +1 (in case of upregulation) or −1 (in case of downregulation).Alv.: alveolospheres; rMCs: resident mesenchymal cells; TG: transgenic; WT: wild type.

Figure 5 .
Figure 5. Treatment with recombinant IL-1β impacts alveolosphere morphology and differentiation.(a) Experimental design for co-culturing WT AEC2s with WT rMCs in the presence or absence of IL-1β.(b) Tile scans of representative wells on day 16.The dashed boxes are magnified in the right panels.(c,d) Quantification of CFE and alveolosphere diameter.(e) Representative maximum intensity projection images of individual organoids from the indicated conditions stained for HOPX, SFTPC, and KRT8.(f) Quantification of the ratio of the KRT8 signal to alveolosphere area.(c,d) Each dot represents one culture well; (f) each dot represents one organoid.* p < 0.05; **** p < 0.0001; ns: not significant.CFE: colony-forming efficiency.Scale bars: (b) 1 mm; (e) 50 µm.

Figure 5 .
Figure 5. Treatment with recombinant IL-1β impacts alveolosphere morphology and differentiation.(a) Experimental design for co-culturing WT AEC2s with WT rMCs in the presence or absence of IL-1β.(b) Tile scans of representative wells on day 16.The dashed boxes are magnified in the right panels.(c,d) Quantification of CFE and alveolosphere diameter.(e) Representative maximum intensity projection images of individual organoids from the indicated conditions stained for HOPX, SFTPC, and KRT8.(f) Quantification of the ratio of the KRT8 signal to alveolosphere area.(c,d) Each dot represents one culture well; (f) each dot represents one organoid.* p < 0.05; **** p < 0.0001; ns: not significant.CFE: colony-forming efficiency.Scale bars: (b) 1 mm; (e) 50 µm.